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===HZAU-China 2022===
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==HZAU-China 2023==
  
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==Design and Expectation==
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===Design and Expectation===
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<p>We hope to induce <i>E.coli</i> BL21 (DE3)(with His-tag)to produce ClyR by IPTG and verify its lysis activity to S. mutans.</p>
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<p>We hope to induce <i>E. coli</i> BL21 (DE3) (with His-tag) to produce ClyR by IPTG and verify its bactericidal activity to <i>S. mutans</i>.</p>
  
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===Materials and Methods===
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==Materials and Methods==
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<p>1. Expression and Purification</p>
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<p>Expression and Purification</p>
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<p>Plasmid pET-28a(+)-ClyR (with His-tag) is transformed to <i>E. coli</i> BL21 (DE3). The <i>E. coli</i> strain is cultured in the LB medium containing 50 μg/mL kanamycin. When the optical density of the cultured bacteria was between 0.6 and 0.8, IPTG was added to the final concentration of 0.2 mM, and the bacteria were induced at 16℃ overnight. Purification was performed following the instructions of Ni<sup>2+</sup>-affinity chromatography. The following is the purification result of the protein.</P>
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<p>Plasmid pET-28a(+)-ClyR (with His-tag) is transformed to <i>E.coli</i> BL21(DE3). The <i>E.coli</i> strain is cultured in the LB medium containing 50 μg/mL kanamycin. When the optical density of the cultured bacteria was between 0.6 and 0.8, IPTG was added to the final concentration of 0.2 mM, and the bacteria were induced at 16℃ overnight.Purification was performed following the instructions of Ni<sup>2+</sup>-affinity chromatography. The following is the purification result of the protein.</P>
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<center><img src="https://static.igem.wiki/teams/4645/wiki/wet-lab/clyr/clyr.png" style="width:40%; "></center>
 
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<center><b>   Figure 1.</b> SDS-PAGE analysis of ClyR </center>
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<center><b>Figure 1. </b> SDS-PAGE analysis of ClyR.</center>
 
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<p>2. Verification of bactericidal activity</p>
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<p> To determine the dose dependence and time dependence of ClyR' s bactericidal activity, <i>S. mutans</i> UA159 was resuspended to an optical density (OD<sub>600</sub>) of ~0.8 using PBS buffer. In a 96-well plate, we sequentially added 35μL of ClyR solution at different concentrations and 165μL of bacterial suspension. The OD<sub>600</sub> was continuously measured using a Synergy H1 microplate reader under constant shaking at 37℃ for 1 hour. The experimental results are shown in the figure below. </p>
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<center><img src="https://static.igem.wiki/teams/4645/wiki/wet-lab/clyr/clyr2.jpg" style="width:60%; "></center>
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<center><b>Figure 2. </b>Variation curve of OD<sub>600</sub> of <i>S.mutans</i> UA159 treated with different ClyR concentrations. </center>
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<p>As shown in the above figure, ClyR demonstrates a certain level of bactericidal activity, which becomes increasingly significant as its concentration increases.</p>

Latest revision as of 12:03, 12 October 2023


Chimeric lysin ClyR with activity against Streptococcus mutans.

ClyR is a chimeric lysin with extended streptococcal host range and has been demonstrated high lytic activity against Streptococcus mutans. It’s composed of a catalytic domain from PlyC and a cell-wall binding domain from PlySs2.


Usage and Biology

This chimeric lysin can bind to S. mutans by a cell-wall binding domain and then lysis its cell wall from outside by a catalytic domain. ClyR has no activity for Gram-negative bacteria and can be expressed in Escherichia coli. With the guidance of appropriate signal peptide, ClyR can be secreted to the outside of the cells, killing S. mutans in the environment.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 757
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 121
    Illegal AgeI site found at 205
    Illegal AgeI site found at 520
    Illegal AgeI site found at 703
  • 1000
    COMPATIBLE WITH RFC[1000]

Functional Parameters

To obtain the ClyR protein, we transferred pET-28a(+)-ClyR (with His-tag) to E. coli BL21(DE3). The E. coli strain was cultured to OD600~0.6, induced with 0.2 mM IPTG, and allowed to grow overnight at 16 ℃. The harvested bacteria were resuspended with a Binding buffer containing 10 mM imidazole, and then the cell was lysis by ultrasonication. Purification was performed following the instructions of Ni-NTA SefinoseTM Resin (Sangon Biotech, Shanghai, China). By SDS-PAGE analysis, we can demonstrate that ClyR can be expressed as a soluble protein in E. coli at the condition described above (Figure 1.). Purified ClyR was desalted and concentrated by Amicon® Ultra-4 Centrifugal Filter Units (Millipore). After quantitation by Bradford assay, the ClyR solution was stored at -20 ℃.

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Figure 1. SDS-PAGE analysis of ClyR expression.


S. mutans UA159 was used as a test strain to verify the activity of purified ClyR protein. Overnight cultured S. mutans UA159 was resuspended in PBS and then treated with 120 μg/mL ClyR. The same volume of PBS was added to another tube as control. After shaking 1 h at 37℃, a significant decrease of turbidity was observed in the ClyR treated group (Figure 2).


Figure 2. Turbidity change of ClyR treated S. mutans UA159.

To determine the dose-dependent and time-dependent lytic activity of ClyR, PBS resuspended S. mutans UA159 was adjusted to an OD600 of ~0.8. In the 96-well plates, 20 μl ClyR solution at different concentrations and 140 μl cell suspension were added in each well. The Synergy H1 microplate reader was used to measure the drop of OD600 at 37 ℃ for 1 h. The results obtain from triple independent experiments are shown below (Figure 3).


Figure 3. Dose-dependent and time-dependent lytic activity of ClyR. A. Variation curve of OD600 of S. mutans UA159 treated with different ClyR concentrations. B. Decrease in OD600 of S. mutans UA159 treated with different concentrations ClyR. The error bars indicate standard error (SEM) of three independent replications.


Besides, we also detected the viable counts of ClyR treated S. mutans. PBS resuspended S. mutans UA159 was treated with ClyR solution in 2 ml EP tubes for 10 min. At the end of the reaction, serial dilutions of each tube were plated on BHI agar and incubated at 37 ℃ overnight. The results obtain from triple independent experiments are shown in Figure 4.


Figure 4. Viable counts of ClyR treated S. mutans. The error bars are obtained from three independent experiments.



Reference

[1] Yang, Hang, et al. "A chimeolysin with extended-spectrum streptococcal host range found by an induced lysis-based rapid screening method." Scientific reports 5 (2015): 17257.
[2] Yang, Hang, et al. "Antibiofilm activities of a novel chimeolysin against Streptococcus mutans under physiological and cariogenic conditions." Antimicrobial agents and chemotherapy 60.12 (2016): 7436-7443.
[3] Xu, Jingjing, et al. "Activity of the chimeric lysin ClyR against common Gram-positive oral microbes and its anticaries efficacy in rat models." Viruses 10.7 (2018): 380.


HZAU-China 2023

Design and Expectation

We hope to induce E. coli BL21 (DE3) (with His-tag) to produce ClyR by IPTG and verify its bactericidal activity to S. mutans.

Materials and Methods

1. Expression and Purification

Plasmid pET-28a(+)-ClyR (with His-tag) is transformed to E. coli BL21 (DE3). The E. coli strain is cultured in the LB medium containing 50 μg/mL kanamycin. When the optical density of the cultured bacteria was between 0.6 and 0.8, IPTG was added to the final concentration of 0.2 mM, and the bacteria were induced at 16℃ overnight. Purification was performed following the instructions of Ni2+-affinity chromatography. The following is the purification result of the protein.


无标题文档


Figure 1. SDS-PAGE analysis of ClyR.

2. Verification of bactericidal activity

To determine the dose dependence and time dependence of ClyR' s bactericidal activity, S. mutans UA159 was resuspended to an optical density (OD600) of ~0.8 using PBS buffer. In a 96-well plate, we sequentially added 35μL of ClyR solution at different concentrations and 165μL of bacterial suspension. The OD600 was continuously measured using a Synergy H1 microplate reader under constant shaking at 37℃ for 1 hour. The experimental results are shown in the figure below.


无标题文档

Figure 2. Variation curve of OD600 of S.mutans UA159 treated with different ClyR concentrations.


As shown in the above figure, ClyR demonstrates a certain level of bactericidal activity, which becomes increasingly significant as its concentration increases.